利用銅沉積技術開發應用於多工細菌檢測之高靈敏度側層流免疫測定試紙元件

Abstract

本研究使用銅沉積訊號放大技術(Copper deposition-induced signal amplification) 應用於金黃色葡萄球菌與綠膿桿菌之側層流免疫測定法(Lateral Flow Imunoassay; LFIA)檢測，並結合可攜帶式光強度偵測儀整合出多工細菌檢測系統。其中本研究測定方法為使用側層流型態完成酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay; ELISA)反應，並且使用傳統金奈米粒子作為目標物辨識依據，側層流反應完成後以硫酸銅及抗壞血酸之還原反應進行訊號放大，用於金黃色葡萄球菌之Protein A偵測及用於綠膿桿菌之Exotoxin A之偵測。為了驗證該訊號放大方法，本研究利用量測光強度來定量目標物濃度，量測結果顯示具顯著放大效果，最後利用光強度偵測晶片結合Arduino UNO所開發之可攜帶式光強度偵測儀對檢測結果作分析與數據呈現。達到定點照護之多工檢測目的。本研究之多工細菌檢測系統，可以於25分鐘內完成細菌偵測。針對金黃色葡萄球菌之Protein A與綠膿桿菌之Exotoxin A進行訊號放大後其最低偵測濃度分別為3 ng/mL與10 ng/mL，與未放大前的偵測強度相差1個數量級。本系統可以在同一時間內偵測兩種細菌感染，並且相較於傳統快篩系統具有更好的靈敏性(sensitivity)。期望未來能結合更多種不同LFIA結構與更換不同放射波長的生物探針，對於更多細菌之生物標記物進行檢測，並擁有更好的檢測靈敏性與特異性。

In this study, the copper deposition signal amplification technology was applied to the lateral laminar flow immunoassay detection of Staphylococcus aureus and Pseudomonas aeruginosa, and combined with a portable light intensity detector to integrate a multiplex bacteria detection system. The measurement method in this study is to use the lateral laminar flow pattern to complete the enzyme-binding immunosorbent analysis reaction, and use traditional gold nanoparticles as the basis for target identification. After the lateral laminar flow reaction is completed, the reduction reaction of copper sulfate and ascorbic acid is used to signal Zoom in for the detection of Protein A of Staphylococcus aureus and the detection of Exotoxin A of Pseudomonas aeruginosa. In order to verify the signal amplification method, this study uses the measured light intensity to quantify the concentration of the target. The measurement result shows a significant amplification effect. Finally, the light intensity detection chip combined with the portable light intensity detector developed by Arduino UNO Analyze the test results and present the data. To achieve the purpose of multi-work detection of designated care. The multi-tasking bacteria detection system in this study can complete bacteria detection within 25 minutes. After signal amplification of Protein A of Staphylococcus aureus and Exotoxin A of Pseudomonas aeruginosa, the lowest detection concentrations are 3 ng/mL and 10 ng/mL, respectively, which are an order of magnitude difference from the detection intensity before unamplification. This system can detect two bacterial infections at the same time, and has better sensitivity than traditional fast screening systems. It is hoped that in the future, it will be able to combine more different LFIA structures and replace bio-probes with different emission wavelengths to detect more bacterial biomarkers, and have better detection sensitivity and specificity.